Dissecting the role of mycobacterial cell envelope components and DNA in leprosy reactions

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Leprosy, a chronic infectious disease caused by the bacterium Mycobacterium leprae, remains a public health threat in developing countries, including Brazil, where more than 30,000 new cases are reported annually. A major health concern for individuals suffering from leprosy is that the disease principally affects the skin and the peripheral nervous system, leading to sensorial impairment and permanent disabilities. Indeed, acute inflammatory reactive leprosy responses of the immune system to the leprosy bacillus are the leading cause of nerve damage. These episodes can occur in individual patients, long after they are considered cured and termination of anti-leprosy drug treatment. However, our understanding of the physiopathology of leprosy reactions remains limited, and further research is urgently needed if we are to understand the biology underlying this neglected disease. The overall aim of this study is to investigate the molecular basis of reactional episodes in leprosy patients. Our hypothesis is that M. leprae constituents can remain in host tissues for long periods of time, and are responsible for the immune activation that occurs in these patients during reaction. This study will bring together the varied and complementary expertise of investigators from The FIOCRUZ, Oswaldo Cruz Foundation (Brazil) and The University of Birmingham (UK) to investigate the involvement of mycobacterial lipids, cell wall fragments and DNA as triggers of the reactional episodes. The investigators based in Brazil are experts in leprosy, specifically the immune responses involved in the infection. Investigators in the UK are experts in the molecular genetics and biochemistry of the mycobacterial cell wall. The knowledge generated in this study can contribute to more effective treatments and management strategies for leprosy reactions, with a greater impact on the quality of life, not just in Brazil but globally, given that leprosy affects many developing countries.The main objectives of the study are:Objective I: The UK Team (University of Birmingham, UoB) will define diagnostic lipid and cell envelope component profiles of standard M. leprae, surrogate Mycobacterium haemophilum and leprosy biopsy material and assess the persistence of these compounds in biopsies from leprosy patients who have experienced a reaction at least two years after the conclusion of anti-leprosy treatment.Objective II: The Brazil Team (FIOCRUZ) will investigate the levels of M. leprae DNA and the involvement of the inflammasome pathways by analysing markers of inflammasome activation in leprosy biopsies of reactional lesions.Objective III: To use well-established extraction and purification protocols at UoB to provide specific cell envelope components for further studies in Objective IV by collaborators at FIOCRUZ (Brazil Team). This will include M. leprae components (e.g. PGL, PDIM, mycolic acids, muropeptides), including available previously purified material by the UK team and through BEI Resources (e.g. whole cells, LAM). Since, leprosy bacilli are uncultivable, M. haemophilum, a phylogenetically related surrogate of M. leprae, will also be used as a source of structurally-related M. leprae-type lipids and LAM for bulk purification. Truncated versions of M. haemophilum PGL and LAM will also be prepared. Objective IV: The Brazil Team (FIOCRUZ) will investigate which mycobacterial components identified in Objectives I and III are bioactive in inducing inflammatory mediators in whole blood samples of reactional patients. The most promising immune active components will be used to investigate their capacity to activate inflammasome pathways in in vitro differentiated macrophages and dendritic cells. Truncated versions of the active molecules will also be tested to map their functional moieties.